Detection of Subclinical Rejection in Pediatric Kidney Transplantation: Current and Future Practices.

INTRODUCTION: The successes in the field of pediatric kidney transplantation over the past 60 years have been extraordinary. Year over year, there have been significant improvements in short-term graft survival. However, improvements in longer-term outcomes have been much less apparent. One important contributor has been the phenomenon of low-level rejection in the absence of clinical manifestations-so-called subclinical rejection (SCR). METHODS: Traditionally, rejection has been diagnosed by changes in clinical parameters, including but not limited to serum creatinine and proteinuria. This review examines the shortcomings of this approach, the effects of SCR on kidney allograft outcome, the benefits and drawbacks of surveillance biopsies to identify SCR, and new urine and blood biomarkers that define the presence or absence of SCR. RESULTS: Serum creatinine is an unreliable index of SCR. Surveillance biopsies are the method most utilized to detect SCR. However, these have significant drawbacks. New biomarkers show promise. These biomarkers include blood gene expression profiles and donor derived-cell free DNA; urine gene expression profiles; urinary cytokines, chemokines, and metabolomics; and other promising blood and urine tests. CONCLUSION: Specific emphasis is placed on studies carried out in pediatric kidney transplant recipients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03719339.

De novo lupus-like glomerulonephritis after pediatric non-kidney organ transplantation.

BACKGROUND: We propose a novel clinically significant finding, de novo lupus-like glomerulonephritis (DNLLGN), in patients with autoantibodies and kidney abnormalities in pediatric liver transplant (LT) and intestinal inclusive transplants (ITx). METHODS: We describe the clinical, serologic, and histopathologic presentation and kidney outcomes in eight patients from our center found to have DNLLGN on kidney biopsy. RESULTS: Pediatric recipients of non-kidney solid organ transplants developed an unusual de novo immune complex glomerulonephritis with morphologic similarity to lupus nephritis. Six had isolated LT (0.9% of all pediatric LT at our center) and two had ITx (2.1% of all ITx). Five (63%) presented with nephrotic syndrome. Five patients had autoantibodies. Patients underwent kidney biopsy at a mean of 11.5 years in LT and 2.8 years in ITx after the index transplant. Biopsies demonstrated changes similar to focal or diffuse active lupus. Follow-up eGFR at a mean of 6 years after biopsy showed a mean decrease of 30 ml/min/1.73 m2 in all patients (p = 0.11). CONCLUSIONS: DNLLGN has not been previously recognized in this clinical setting, yet 8 kidney biopsies from pediatric recipients of LT and ITx at our center in 25 years demonstrated this finding. DNLLGN appears to be an under-reported phenomenon of clinical significance. A higher resolution version of the Graphical abstract is available as Supplementary information.

An age-independent gene signature for monitoring acute rejection in kidney transplantation.

Acute rejection (AR) remains a significant problem that negatively impacts long-term renal allograft survival. Numerous therapies are used to prevent AR that differ by center and recipient age. This variability confounds diagnostic methods. Methods: To develop an age-independent gene signature for AR effective across a broad array of immunosuppressive regimens, we compiled kidney transplant biopsy (n=1091) and peripheral blood (n=392) gene expression profiles from 12 independent public datasets. After removing genes differentially expressed in pediatric and adult patients, we compared gene expression profiles from biopsy and peripheral blood samples of patients with AR to those who were stable (STA), using Mann-Whitney U Tests with validation in independent testing datasets. We confirmed this signature in pediatric and adult patients (42 AR and 47 STA) from our institutional biorepository. Results: We identified a novel age-independent gene network that identified AR from both kidney and blood samples. We developed a 90-probe set signature targeting 76 genes that differentiated AR from STA and found an 8 gene subset (DIP2C, ENOSF1, FBXO21, KCTD6, PDXDC1, REXO2, HLA-E, and RAB31) that was associated with AR. Conclusion: We used publicly available datasets to create a gene signature of AR that identified AR irrespective of immunosuppression regimen or recipient age. This study highlights a novel model to screen and validate biomarkers across multiple treatment regimens.

Sirolimus and tacrolimus coefficient of variation is associated with rejection, donor-specific antibodies, and nonadherence.

BACKGROUND: Immunosuppression medication nonadherence has been associated with donor-specific antibodies and treatment-refractory rejection. Drug-level monitoring is a practical direct marker for nonadherence, as variations indicate erratic ingestion of medication. We previously reported that high variability in tacrolimus trough levels determined by the percent coefficient of variation (CV %) and standard deviation (SD) were associated with biopsy-proven rejection. We hypothesized that the CV % and SD in patients on a sirolimus/low-dose tacrolimus regimen may associate with self-reported medication nonadherence, rejection and donor-specific antibodies. METHODS: In this pilot feasibility study, we studied 37 biopsies in 23 pediatric renal transplant patients on both sirolimus and tacrolimus immunosuppression; CV %, SD, de novo donor-specific antibodies, rejection, and self-reported adherence were examined. RESULTS: A cut-off sirolimus CV % of 25 maximized the percentage of biopsies correctly classified as rejection (32 of 37, or 86 %, p = 0.001). A cut-off tacrolimus CV % of 31 maximized the percentage of correctly classified biopsies (25 of 37, or 68 %, p = 0.09). Among patients with both high sirolimus and tacrolimus CV %, 67 % developed de novo donor-specific antibodies (p = 0.002) with a DQ predominance and 71 % reported nonadherence (p = 0.05). CONCLUSIONS: In pediatric renal transplantation, sirolimus and tacrolimus CV % is a potential tool for monitoring patients at risk for allograft rejection and donor-specific antibodies secondary to medication nonadherence.

Bortezomib may stabilize pediatric renal transplant recipients with antibody-mediated rejection.

BACKGROUND: Current therapeutic strategies to effectively treat antibody-mediated rejection (AMR) are insufficient. Thus, we aimed to determine the benefit of a therapeutic protocol using bortezomib for refractory C4d + AMR in pediatric kidney transplant patients. METHODS: We examined seven patients with treatment-refractory C4d + AMR. Immunosuppression included antithymocyte globulin or anti-CD25 monoclonal antibody for induction therapy with maintenance corticosteroids, calcineurin inhibitor, and anti-metabolite. Estimated glomerular filtration rate (eGFR) calculated by the Schwartz equation, biopsy findings assessed by 2013 Banff criteria, and human leukocyte antigen (HLA) donor-specific antibodies (DSA) performed using the Luminex single antigen bead assay were monitored pre- and post- bortezomib therapy. RESULTS: Seven patients (86 % male, 86 % with ≥6/8 HLA mismatch, and 14 % with pre-formed DSA) age 5 to 19 (median 15) years developed refractory C4d + AMR between 1 and 145 (median 65) months post-transplantation. All patients tolerated bortezomib. One patient had allograft loss. Of the six patients with surviving grafts (86 %), mean pre-bortezomib eGFR was 42 ml/min/1.73 m(2) and the mean 1 year post-bortezomib eGFR was 53 ml/min/1.73 m(2). Five of seven (71 %) had improvement of histological findings of AMR, C4d staining, and/or acute cellular rejection. Reduction in HLA DSAs was more effective for class I than class II. CONCLUSIONS: Bortezomib appears safe and may correlate with stabilization of eGFR in pediatric kidney transplant patients with refractory C4d + AMR.

A novel treatment regimen for BK viremia.

BACKGROUND: BK viremia, a prerequisite for BK virus nephropathy (BKVN), affects 5% to 16% of pediatric renal transplant recipients (PRTR). We evaluated the safety and efficacy of a novel approach to treating BK viremia using fluoroquinolones and leflunomide in PRTR. METHODS: We studied 230 PRTR at Mattel Children's Hospital, UCLA, who underwent renal transplantation between January 2003 and October 2010. Nineteen patients were found to have BK viremia. Ciprofloxacin was started when the BK viral load was greater than 625 copies/mL, and patients were switched to leflunomide if BK viral load did not decrease after 2 months of ciprofloxacin therapy. All patients underwent transplant kidney biopsy, and their estimated glomerular filtration rate (eGFR) and BK PCR was measured serially. The side effects of ciprofloxacin and leflunomide were recorded in each patient. RESULTS: There was a significant decrease in BK viral load in patients treated with ciprofloxacin and leflunomide (P<0.001) with only a small reduction in immunosuppression. BK viremia was associated with a significantly decreased eGFR (P<0.001), and treatment with ciprofloxacin and leflunomide was associated with improved eGFR (P<0.001). This approach resulted in a BKVN rate of only 1%. CONCLUSIONS: This analysis demonstrates for the first time that, used in a stepwise fashion, ciprofloxacin and leflunomide are effective and safe treatments for BK viremia in PRTR.

Monitoring nonadherence and acute rejection with variation in blood immunosuppressant levels in pediatric renal transplantation.

BACKGROUND: Acute rejection associated with medication nonadherence is a major cause of allograft loss in pediatric kidney transplant patients. There is currently no reliable method to detect medication nonadherence and prevent allograft rejection. METHODS: In 46 pediatric patients who underwent renal transplantation between 2002 and 2003, the variation of serum drug levels was studied as a potential objective tool to monitor medication nonadherence. Tacrolimus (TAC) and mycophenolic acid (MPA) trough levels were measured from 1 to 12 months posttransplant, and standard deviation (SD) and percent coefficient of variation (CV%) were calculated. Because SD increased as mean trough levels rose, CV% (CV%=SD/mean multiplied by 100%) was used to eliminate this confounding effect. RESULTS: Ten of 46 patients had biopsy-proven rejection. The median TAC CV% was 53.4% in patients with biopsy-proven rejection, which was significantly higher than 30% in those without rejection (P=0.005). Median MPA CV% was 51.9% in patients without rejection and 45.1% in patients with rejection (P=NS). High TAC CV% correlated with increased risk for rejection, whereas MPA CV% did not. CONCLUSION: The TAC CV% seems to be a useful and superior marker, compared with SD alone, for assessing medication nonadherence and the possibility of allograft rejection in pediatric renal transplantation.

Acute rejection associated with donor-specific anti-MICA antibody in a highly sensitized pediatric renal transplant recipient.

Allograft rejection in HLA identical transplant recipients and in patients without detectable donor-specific anti-HLA antibodies has lead to the identification of non-HLA antigens as targets of the alloimmune response. MICA antigen has been recognized as an important non-HLA target in renal transplantation. Recent studies have shown that anti-MICA antibodies are associated with acute renal allograft rejection and failure. Current cross match procedures using donor lymphocytes fail to detect MICA antibodies. Transplant candidates are not routinely tested for pre-sensitization to MICA antigens nor are transplant donors typed for MICA alleles. Optimal classification and treatment of acute rejection associated with MICA antibody remains unknown. In this case report, we are the first to describe the clinical course and treatment of donor-specific MICA antibody associated with both Banff type II A ACR and AMR in a highly sensitized pediatric renal re-transplant recipient. This case also emphasizes the importance of pre-transplant screening for donor-specific MICA antibody especially in highly sensitized renal transplant patients.

Significance of intragraft CD138+ lymphocytes and p-S6RP in pediatric kidney transplant biopsies.

BACKGROUND: We have previously shown that intragraft CD20+ B cells are associated with acute cellular rejection (ACR) and allograft loss. Phosphorylation of S6 ribosomal protein, a downstream target of the PI3K/Akt/mTOR pathway, promotes growth and proliferation of cells and could identify metabolically active cells such as alloantibody secreting plasma cells. Because CD20+ lymphocytes can differentiate into CD138+ plasma cells, we aimed to identify functionally active plasma cells by using intragraft CD138 quantification and p-S6RP staining and correlate these results with allograft rejection, function, and survival. METHODS: We examined 46 renal transplant biopsies from 32 pediatric patients who were biopsied for clinical suspicion of rejection. Immunohistochemical staining for C4d, CD20, CD138, and p-S6RP was performed. Patient creatinine clearance and graft status was followed up postbiopsy. RESULTS: Patients with greater than or equal to six CD138+ cells/high power field (hpf) had worse graft survival with a hazard ratio of 3.4 (95% CI 1.3-9.2) 2 years postbiopsy compared with those with 0 to 5 cells/hpf (P=0.016). CD138+ cells were stained for p-S6RP, indicating functionally active plasma cells. They were associated with ACR (P=0.004) and deteriorating graft function (R=0.22, P=0.001). Intragraft CD20+ and CD138+ cells found together in ACR were associated with poorer graft survival than either marker alone, hazard ratio 1.5 (95% CI 1.1-2.2, P=0.01). CONCLUSIONS: A threshold of greater than or equal to six CD138+ metabolically active plasma cells per hpf, coexisting with CD20+ B cells, was associated with poor allograft function and survival. This may represent an additional antibody-mediated process present in the setting of ACR and could play an important role in characterization and treatment of transplant rejection.

Trabecular bone volume and osteoprotegerin expression in uremic rats given high calcium.

Calcium (Ca)-containing phosphate binders have been recommended for the treatment of hyperphosphatemia in children with chronic kidney disease. To study the effects of high Ca levels on trabecular bone volume (BV) and osteoprotegerin (OPG) expression in uremic young rats, a model of marked overcorrection of secondary hyperparathyroidism was created by providing a diet of high Ca to 5/6 nephrectomized young rats (Nx-Ca) for 4 weeks. The results of chondrocyte proliferation and apoptosis, osteoclastic activity, OPG expression and BV were compared among intact rats given the control diet, intact rats given a high Ca diet and 5/6 nephrectomized rats given the control diet (Nx-Control) and the high Ca diet (Nx-Ca). Ionized Ca levels were higher and parathyroid hormone levels were lower in Nx-Ca rats than in the other groups. Final weight, final length and final tibial length of Nx-Ca rats were significantly less than those of the other groups, although the length gain did not differ among the groups. The hypertrophic zone width was markedly enlarged in Nx-Ca rats. Chondrocyte proliferation rates did not differ among the groups, whereas osteoclastic activity was decreased in Nx-Ca rats compared with the Nx-Control animals. The OPG expression and BV were increased in Nx-Ca rats compared with the Nx-Control rats. Increased BV should improve bone strength, whereas disturbance of osteoclastogenesis interferes with bone remodeling. Bone quality has yet to be determined in high Ca-fed uremic young rats.

CD20+ lymphocytes in renal allografts are associated with poor graft survival in pediatric patients.

BACKGROUND: The presence of CD20+ lymphocyte renal allograft infiltrates has been associated with steroid-resistant rejection and poor graft survival. We quantified the number of CD20+ lymphocytes in renal allograft biopsies and correlated the results with graft survival. We also determined the relationships between CD20+ lymphocytes and acute cellular rejection versus antibody-mediated rejection. METHODS: We examined 45 biopsy samples from 31 pediatric patients biopsied for suspicion of rejection from November 2001 to November 2004. Immunohistochemical staining for CD20 and C4d was performed on all biopsies; CD20+ cell density per high-power field (hpf) was determined for each core. Patient graft status was followed postbiopsy and documented for graft survival or failure using the cutoff date of December 31, 2005. RESULTS: Patients with 2-10 and 11-100 CD20+ cells/hpf had worse graft survival in Kaplan-Meier analysis with a hazard ratio 4.56 (CI 1.07-19.35) two years postbiopsy compared to those with 0-1 cells/hpf (P = 0.02). The presence of CD20+ lymphocytes was significantly associated with acute cellular rejection (P = 0.0001) and not associated with antibody-mediated rejection (P = 0.16). Receiver-operating curve analysis confirmed > or =3 cells/hpf correlating with acute cellular rejection, yielding sensitivity 90% and specificity 76%. CONCLUSIONS: This study shows a significant 4.5-fold risk of graft failure at two years postbiopsy with presence of > or =2 CD20+ cells/hpf. Moreover, > or =3 CD20+ lymphocytes were highly associated with acute cellular rejection. They may be functioning as professional antigen-presenting cells in the graft. In steroid-refractory cellular rejections, therapies that target B cells may prolong graft survival.

Laparoscopic versus open renal procurement for pediatric recipients of living donor renal transplantation.

Despite reports demonstrating the safety of laparoscopic donor nephrectomy (LDN) for pediatric recipients of renal transplants, recent evidence has challenged using LDN for recipients 5 years of age or younger. We retrospectively reviewed the records of all pediatric recipients of living donor renal transplants from September 2000 through August 2004. We compared those who received allografts recovered by LDN (n = 34) with those recovered by open donor nephrectomy (ODN, n = 26). Outcomes of interest included operative complications, postoperative renal function, the incidence of delayed graft function or episodes of acute rejection and long-term graft function. Donor and recipient demographic data were similar for the LDN and ODN groups. Serum creatinine and calculated creatinine clearance were not significantly different between groups both in the early postoperative period and at long-term follow-up (p > 0.142). Rates of delayed graft function and acute rejection did not differ between groups. Among recipients aged 5 years old or younger stratified by donor technique (9 LDN, 5 ODN recipients), no difference was noted in graft outcomes both early and long-term (p > 0.079). At our center, pediatric LDN recipients have graft outcomes comparable to those of ODN recipients. At experienced centers, we recommend continued use of LDN for pediatric recipients of all ages.

Growth hormone and the skeleton in pediatric renal allograft recipients.

Recombinant human growth hormone has been utilized to augment linear growth in pediatric renal allograft recipients. The skeletal changes that accompany growth hormone therapy have not been described in children. Thus, 23 stable prepubertal pediatric kidney recipients, aged 10 +/- 3 years, with a mean transplant time of 3.4 +/- 2.5 years and histological findings of normal bone formation and adynamic bone on bone biopsies were prospectively randomized into two groups. These comprised a treated group that received 12 months of growth hormone and a control group that did not receive any treatment. Anthropometric measurements and blood for serum calcium, phosphorus, parathyroid hormone (PTH), osteocalcin, and insulin-like growth factor-I (IGF-I) were obtained every 3 months. Measurements of bone mass by dual-energy X-ray absorptiometry were performed at the beginning and end of the study period. All patients underwent an initial and final bone biopsy procedure after double tetracycline labeling. Annual growth velocity increased and standard deviation scores for height improved in the treated group. Serum IGF-I levels increased in the treated group and the increase was evident in patients with normal bone formation who received growth hormone but not in patients with adynamic bone. Serum calcium, phosphorus, osteocalcin, and PTH levels did not differ between the treated and control groups. Bone mass did not change in the treated group, but declined after 12 months in the control group. Bone formation rates did not increase with growth hormone treatment. Thus, growth hormone therapy improves linear growth and maintains bone mass, but does not favorably affect bone formation rates in stable pediatric renal allograft recipients.